Proteins play an important role in today's medical portfolio. Expression systems for the production of recombinant polypeptides are well-known in the state of the art. Polypeptides for use in pharmaceutical applications are mainly produced in prokaryotic cells, such as E. coli, and mammalian cells such as CHO cells, NSO cells, Sp2/0 cells, COS cells, HEK cells, BHK cells, PER.C6® cells, and the like.
For human application every pharmaceutical substance has to meet distinct criteria. To ensure the safety of biopharmaceutical agents to humans, for example, nucleic acids, viruses, and host cell proteins, which would cause severe harm, have to be removed. To meet the regulatory specification one or more purification steps have to follow the manufacturing process. Among other, purity, throughput, and yield play an important role in determining an appropriate purification process.
Different methods are well established and widespread used for protein purification, such as affinity chromatography with microbial proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (sulfopropyl or carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode ion exchange), thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with Ni(II)- and Cu(II)-affinity material), size exclusion chromatography, and electrophoretical methods (such as gel electrophoresis, capillary electrophoresis) (see e.g. Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75 (1998) 93-102).
Mateo, C., et al. report about the affinity chromatography of polyhistidine-tagged enzymes (J. Chrom. 915 (2001) 97-106). In WO 02/37100 novel applications of nickel nitrilotriacetic acid (NI-NTA) resin are reported. Methods and kits for purifying his-tagged proteins are reported in WO 2005/035092. In WO 98/06739 a method for the purification of recombinant proteins is reported.
Affinity purification methods involving amino acid mimetics as elution agents are reported in WO 94/07912. In US 2004/0152076 nucleic acid separation using immobilized metal affinity chromatography. A process for the purification of factor VIII is reported in U.S. Pat. No. 6,005,082. In US 2007/0037966 a hydrophobic interaction chromatography purification of factor VII polypeptides is reported.